Preparation and properties of a cross‐linked complex between ferredoxin–NADP+ reductase and flavodoxin

Abstract

The electrostatically stabilized complex between Anabaena variabilis ferredoxin–NADP⁺ reductase and Azotobacter vinelandii flavodoxin has been covalently cross‐linked by treatment with 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide. The covalent complex exhibits a molecular mass and FMN/FAD content consistent with that expected for a 1:1 stoichiometry of the two flavoproteins. Immunochemical cross‐reactivity is exhibited by the covalent complex with rabbit antisera prepared separately against each protein. The complex retains NADPH–ferricyanide diaphorase activity although the K_m for ferricyanide is increased twofold and the turnover number is decreased by a factor of two when compared to native reductase. NADPH–cytochrome‐c reductase activity of the complex is observed at a level that is quite similar to that determined at saturating concentrations of flavodoxin, while it is only 1–2% of that exhibited by the reductase in the presence of ferredoxin. No stimulation of cytochrome‐c reductase activity is observed on adding ferredoxin to the cross‐linked complex. Stopped‐flow data show that covalent cross‐linking of the flavodoxin to the reductase reduces the rate of electron transfer from its semiquinone form to cytochrome c by a factor of 60. Anaerobic titrations of the reduced complex with NADP⁺ show the semiquinone/quinol couple of the flavodoxin is increased 100 mV relative to the free form and the quinone/quinol couple of complexed ferredoxin– NADP⁺ reductase is increased by only 25 mV, relative to the free protein. Addition of NADPH to the cross‐linked complex reduces the FAD of the reductase as well as the FMN moiety of flavodoxin to a mixture of somiquinone and quinol forms.